Type IV secretion systems (T4SS) are used by many medically-important Gram-negative bacterial pathogens to deliver antibiotic resistant genes among bacterial populations and translocate virulence factors into eukaryotic host cells. This multiprotein protein complex is evolutionarily-conserved among Gram-negative bacterial populations, and consists of a core set of proteins that assemble to span the bacterial envelope as a translocation channel and in many cases an extracellular pilus. Despite their broad medical importance, the molecular mechanism of action and the overall architecture of T4SSs are not well understood. This application seeks to define the ultrastructure of a model T4SS within the cell envelope of Escherichia coli in unprecedented detail. This is afforded by recent discoveries that E. coli minicells, skinny cells and cells digested of their cell walls permit visualization ofcell envelope organelles at unparalleled resolution by cryoelectron tomography (CryoET). The overall hypothesis of this proposal is that T4SSs of Gram-negative bacteria are composed of two subassemblies, the inner and outer membrane complexes, whose activities are coordinated to mediate substrate transfer across the bacterial cell envelope. In Aim 1, I will determine structures of outer membrane complexes produced on intact cells and define the genetic requirements for assembly of this substructure by analysis of cells producing T4SSs lacking one or more of the machine subunits. In Aim 2, I will visualize the structure of the inner membrane complex both in the absence and presence of the outer membrane complex and with and without docked substrate. I will also develop a novel translocation assay to test for functionality of the inner membrane complex in various genetic contexts. Together, my studies will utilize complementary molecular genetic, biochemical and ultrastructural approaches, the latter capitalizing on the availability of a state-of-the-art cryoEM/ET facility and expertise in high-resolution structural approaches that are available at my institution. The completion of this proposal will provide important insight into the structure and function of a model T4SS. My findings will form a foundation for future work aimed at suppressing the action of these medically-important translocation systems.